Interaction of inhibitors of the vacuolar H(+)-ATPase with the transmembrane Vo-sector.

The macrolide antibiotic concanamycin A and a designed derivative of 5-(2-indolyl)-2,4-pentadienamide (INDOL0) are potent inhibitors of vacuolar H(+)-ATPases, with IC(50) values in the low and medium nanomolar range, respectively. Interaction of these V-ATPase inhibitors with spin-labeled subunit c in the transmembrane V(o)-sector of the ATPase was studied by using the transport-active 16-kDa proteolipid analogue of subunit c from the hepatopancreas of Nephrops norvegicus. Analogous experiments were also performed with vacuolar membranes from Saccharomyces cerevisiae. Membranous preparations of the Nephrops 16-kDa proteolipid were spin-labeled either on the unique cysteine C54, with a nitroxyl maleimide, or on the functionally essential glutamate E140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). These residues were previously demonstrated to be accessible to lipid. Interaction of the inhibitors with these lipid-exposed residues was studied by using both conventional and saturation transfer EPR spectroscopy. Immobilization of the spin-labeled residues by the inhibitors was observed on both the nanosecond and microsecond time scales. The perturbation by INDOL0 was mostly greater than that by concanamycin A. Qualitatively similar but quantitatively greater effects were obtained with the same spin-label reagents and vacuolar membranes in which the Nephrops 16-kDa proteolipid was expressed in place of the native vma3p proteolipid of yeast. The spin-label immobilization corresponds to a direct interaction of the inhibitors with these intramembranous sites on the protein. A mutational analysis on transmembrane segment 4 known to give resistance to concanamycin A also gave partial resistance to INDOL0. The results are consistent with transmembrane segments 2 and 4 of the 16-kDa putative four-helix bundle, and particularly the functionally essential protonation locus, being involved in the inhibitor binding sites. Inhibition of proton transport may also involve immobilization of the overall rotation of the proteolipid subunit assembly.

Dicyclohexylcarbodiimide inhibits proton pumping in ubiquinol:cytochrome c oxidoreductase of Rhodobacter sphaeroides and binds to aspartate-187 of cytochrome b.

In recent studies we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in ubiquinol:cytochrome c oxidoreductase (cytochrome bc1 complex) from yeast mitochondria where it was bound to aspartate-160 of cytochrome b. In the current study, we report that DCCD and its fluorescent analogue, N-cyclohexyl-N'-[4-(dimethylamino)naphthyl]-carbodiimide (NCD-4), inhibit 50-60% proton pumping in the cytochrome bc1 complex of the bacterium Rhodobacter sphaeroides with a 20% inhibition of electron transfer activity. Radioactive DCCD is bound exclusively to cytochrome b at aspartate-187, which is located at the C-terminal region of the CD loop connecting membrane-spanning helices C and D of cytochrome b. Fluorescent studies with NCD-4 revealed that aspartate-187 is located in a mildly hydrophobic pocket in the bc1 complex at a distance of 2-3 A from the surface of the membrane.

Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatized aspartyl-61.

Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two alpha-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein in the membrane. The relevance of the structure in solution to the native structure was demonstrated. Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution. Further, the protein purified from the Ile28-->Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution. This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm. Subunit c in wild-type E. coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified. DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances. The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample. The distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca. 12 A) to the derivatized Asp61 in helix-2. A model for the interaction of helices in the NCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of < 10-20 A derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2-5 A) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein.

Inhibition of (Ca2+ + Mg2+)-ATPase by carbodiimides. A structure-activity study.

Nine hydrophobic carbodiimides were synthesized and their chemical reactivities (towards acetic acid) and inhibitory capacities (towards the (Ca2+ + Mg2+)-ATPase were measured. No correlation between chemical reactivity and inhibitory efficacy emerges, but a significant effect of molecular bulk on reactivity towards the calcium-protectable carboxyl groups of the ATPase is noted: methyl-substituted compounds inhibit the enzyme in the presence of Ca2+, while aryl- or cyclohexyl-substituted compounds do not inactivate in the presence of Ca2+.